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1.
Front Med (Lausanne) ; 9: 865035, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35721049

RESUMO

Background and Aims: Severe discomfort during an upper gastrointestinal endoscopy (UGE) is often a stressful experience for patients undergoing the procedure. An increasing number of studies have shown that acupuncture may reduce discomfort during UGE. A systematic review in 2004 investigated the effect of acupuncture for gastrointestinal endoscopy, but these data have not been recently reviewed. Therefore, this study was conducted to evaluate the current evidence and provide up-to-date knowledge for clinical decision-making. Methods: Nine databases were searched from inception to June 2021. Eligible randomized controlled trials (RCTs) were included. The outcome data were synthesized where necessary, and risks of bias of included studies were assessed using RevMan V.5.3. Results: Twenty-three eligible RCTs with 3,349 patients were identified. It was found that acupuncture plus topical pharyngeal anesthesia with lidocaine hydrochloride (TPALH) resulted in greater improvements regarding visual analog scale (VAS) scores and the incidence of nausea and vomiting (INV) when compared with TPALH alone. These results were consistent among studies of manual acupuncture, electroacupuncture, auricular-plaster, superficial needle (SFN) and acupressure. In the meta-analysis, SFN plus TPALH showed significant improvement of VAS scores compared to sham SFN plus TPALH (MD -1.11, 95% CI -1.52 to -0.70, P < 0.00001). Most of included studies did not report any side effects in their findings, and were of medium-to-high risk of bias. Conclusion: Acupuncture, as adjunctive therapy to TPA, may result in less patient discomfort than TPA alone. Findings from this review should be interpreted with caution due to the high heterogeneity identified. There is low-quality evidence supporting the use of acupuncture over sham. More rigorously designed RCTs are needed to inform clinical decision-making. Systematic Review Registration: PROSPERO [CRD42014008966].

2.
Nat Commun ; 13(1): 3410, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35701497

RESUMO

Ribosome associated complex (RAC), an obligate heterodimer of HSP40 and HSP70 (Zuo1 and Ssz1 in yeast), is conserved in eukaryotes and functions as co-chaperone for another HSP70 (Ssb1/2 in yeast) to facilitate co-translational folding of nascent polypeptides. Many mechanistic details, such as the coordination of one HSP40 with two HSP70s and the dynamic interplay between RAC-Ssb and growing nascent chains, remain unclear. Here, we report three sets of structures of RAC-containing ribosomal complexes isolated from Saccharomyces cerevisiae. Structural analyses indicate that RAC on the nascent-chain-free ribosome is in an autoinhibited conformation, and in the presence of a nascent chain at the peptide tunnel exit (PTE), RAC undergoes large-scale structural remodeling to make Zuo1 J-Domain more accessible to Ssb. Our data also suggest a role of Zuo1 in orienting Ssb-SBD proximal to the PTE for easy capture of the substrate. Altogether, in accordance with previous data, our work suggests a sequence of structural remodeling events for RAC-Ssb during co-translational folding, triggered by the binding and passage of growing nascent chain from one to another.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
3.
Transl Cancer Res ; 11(5): 1188-1194, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35706819

RESUMO

Background: This study sought to compare the consistency of the epidermal growth factor receptor (EGFR) gene mutation detection results in the supernatant of alveolar lavage specimens to the tissue sample results, and the consistency of the blood EGFR gene mutation detection results to the tissue detection results. Methods: In total, 29 patients with non-small cell lung carcinoma (NSCLC) were selected, and their bronchoalveolar lavage fluid (BALF) was collected. The supernatant and precipitate were separated by centrifugation. Deoxyribonucleic acid (DNA) was extracted from the supernatant, and blood and tumor tissues were collected to detect patients' EGFR gene mutation status. Results: Of the 29 enrolled patients, 12 of the 23 tissue-biopsy patients (52.2%) were positive for EGFR mutations, 11 of the 28 blood-test patients (39.2%) were positive for EGFR mutations, and 13 of the 29 cases of the BALF-test patients (44.8%) were positive for EGFR mutations. The most common mutations were the exon 19 deletion mutation and the L858R point mutation. The EGFR gene mutation rate was higher in female, young, non-smoker, and stage IIIB patients (than stage IV patients), but the differences were not statistically significant (all P>0.05). Of the 29 NSCLC patients tested for the EGFR gene mutation, the BALF supernatant and blood results were the same for 27 patients (coincidence rate: 93.10%). Of the 23 of the 29 enrolled patients tested for the EGFR gene mutation, the BALF supernatant and tissue test results were the same for 21 patients (coincidence rate: 91.30%). Further, the blood-test and the tissue test results were the same for 20 patients (coincidence rate: 86.96%). Conclusions: The EGFR gene mutation rate was high in NSCLC patients. The coincidence rate of the EGFR gene mutation detection results between BALF supernatant and tumor tissues was slightly higher than that of the blood and tumor tissue EGFR gene mutation detection results.

4.
Front Nutr ; 9: 910949, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35669075

RESUMO

Background: Although studies suggest that concentrations of serum 25-hydroxyvitamin D (25(OH)D) are lower in individuals with Heart Failure (HF), the beneficial effects of vitamin D supplementation are controversial. Therefore, in this study, we aimed to determine whether there is a causal relationship between serum Vitamin D (VD) levels and HF. Methods: We obtained genetic instruments from the largest available genome-wide association study (GWAS) of European descent for 25(OH)D (443, 734 individuals) to investigate the association with HF (47,309 cases, 930,014 controls), and vice versa. Two-sample bidirectional Mendelian Randomization (MR) analysis was performed to infer the causality. In addition to the primary analysis using inverse variance-weighted (IVW) MR, we applied five additional methods to control for pleiotropy [MR-Egger, weighted median, Maximum-likelihood, MR-robust adjusted profile score (MR-RAPS) and MR-pleiotropy residual sum and outlier (MR-PRESSO)] and compared their respective MR estimates. We also performed a sensitivity analysis to ensure that our results were robust. Results: Mendelian randomized analysis showed that increased serum 25(OH)D was associated with a lower risk of HF in the IVW method (odds ratio [OR] = 0. 81;95%CI, 0.70-0.94, P = 0.006). In the reverse MR analyses, the genetic predisposition to HF was negatively correlated with serum 25(OH)D level (OR = 0. 89;95%CI, (0.82-0.97), P = 0.009). Conclusion: Our study revealed the possible causal role of 25(OH)D on decreasing the risk for HF. Meanwhile, reverse MR analysis suggested that HF may be associated with lower vitamin D levels, it could be the potential implications for dietary recommendations.

5.
Nucleic Acids Res ; 2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35639884

RESUMO

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

6.
Ecol Evol ; 12(5): e8905, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35571753

RESUMO

The exotic plant Spartina alterniflora is expanding rapidly along China's coast regions, seriously threatening native ecosystems. Soil bacteria are important for biogeochemical cycles, including those of carbon, nitrogen, and sulfur, in wetland ecosystems. There is growing evidence that microorganisms are important in case of plant invasion. In the present study, we studied the interlacing area of S. alterniflora and Suaeda heteroptera, selected soil of invaded and non-invaded regions and explored the effect of the composition and diversity of bacterial communities in coastal wetlands. The bacterial community composition of invasive and noninvasive areas was subjected to high-throughput sequencing. In the five areas tested, the main bacterial phyla were Proteobacteria, Bacteroides, and Acidobacteria; the richness of the bacterial community in the soil increased after S. alterniflora invasion, most changes occurred at the genus level. The relative abundances of Desulfobulbus and Sulfurovum were higher in invasive areas than in noninvaded areas. PCA, RDA, and LEfSe analyses found that the S. alterniflora invasion significantly influenced the bacterial community and physicochemical properties of wetland soil. In conclusion, soil microbial community composition was tightly associated with S. alterniflora invasion. This study provide an important scientific basis for further research on the invasion mechanism of S. alterniflora.

7.
Front Cardiovasc Med ; 9: 822150, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35600473

RESUMO

Objective: We report a 3-generation family with SCN5A c.611 C>A rare variant, whose clinical characteristics are dilated cardiomyopathy (DCM) combined with multifocal ectopic Purkinje-related premature contractions (MEPPC). We tried to explain why the same SCN5A variant carriers had different phenotypes. Methods: We collected the clinical data from the family, and followed up this family members. Genetic testing was done for whom DNA samples could be collected. Results: Information was collected from 15 people in this family, 8 of whom had genetic testing. The SCN5A variant was present in all patients of this family, whose clinical features showed DCM combined with MEPPC. The proband's children developed DCM and MEPPC in their childhood. They both carried a SCN5A p.A204E mutation from their mother and a mutation PRKAG2 p.D372N from their father. The son did heart transplant and his heart was both dilated and thickened. The pathology confirmed the presence of glycogen accumulation in the myocardium, which were consistent with the diagnosis of PAKAG2 syndrome. Conclusion: SCN5A c.611 C>A variant was related to DCM combined with MEPPC. This case report is the first to demonstrate that a combination of SCN5A and PRKAG2 mutations can cause DCM plus MEPPC and PRKAG2 Syndrome.

8.
Methods Mol Biol ; 2467: 341-357, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35451782

RESUMO

In this chapter, we discuss the motivation for integrating other types of omics data into genomic prediction methods. We give an overview of literature investigating the performance of omics-enhanced predictions, and highlight potential pitfalls when applying these methods in breeding. We emphasize that the statistical methods available for genomic data can be transferred to the general omics case. However, when using a framework of omic relationship matrices, the standardization of the variables may be more relevant than it is for a genomic relationship matrix based on single-nucleotide polymorphisms.


Assuntos
Genômica , Modelos Genéticos , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas
9.
Sci Adv ; 8(17): eabm2343, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35486720

RESUMO

Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, Photorhabdus virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo-electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.


Assuntos
Photorhabdus , Animais , Microscopia Crioeletrônica , Photorhabdus/metabolismo , Cloreto de Polivinila/metabolismo , Sinais Direcionadores de Proteínas , Virulência
11.
Int J Ophthalmol ; 15(4): 657-660, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35450178

RESUMO

AIM: To achieve a balance between efficiency and cost in the management of medical instrument by the use of micro needle holder. METHODS: In this study, the novel multifunctional use of micro needle holders was performed between 2018 and 2019 at the Department of Ophthalmology in the 4 hospitals in Shaanxi Province. In this innovation, the micro needle holders were initially used as micro forceps to remove sutures, as eye spud to safety extract foreign body from cornea, as ciliary forceps to remove trichiasis, as well as punctal dilator to dilate most small puncta. RESULTS: Using this technique, the medical costs of both procurement and sterilization were cut off in the selected 4 hospitals. The purchase cost has dropped by roughly 50%. The sterilization cost was decreased by about 30%. CONCLUSION: The innovation in the five-in-one multifunctional use of micro needle holders saves the medical costs.

12.
Genes (Basel) ; 13(4)2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35456366

RESUMO

The genus Malus is rich in species and many of its plastid genomes have been released. However, limited resources and few markers are not conducive to the comparison of differences among species and resource identification and evaluation. In this study, the complete chloroplast genome of Malus zhaojiaoensis was studied by NGS sequencing, with a total length of 159998 bp. It consists of four regions, LSC (88,070 bp), IRB (26,359 bp), SSC (19,210 bp) and IRA (26,359 bp). M. zhaojiaoensis cp genome contained a total of 111 genes made up of three classes: 78 coding sequences, 29 tRNA genes, and four rRNA genes. In addition, a total of 91 SSRs and 43 INEs were found in the M. zhaojiaoensis cp genome, which was slightly different from M. baccata and M. hupehensis in number. The analysis of codon usage and RNA editing showed that high-frequency codons tended to end at A/U bases and RNA editing mainly occurred at the second codon. Comparative genome analysis suggested that the cp genomes of eight Malus species had higher overall similarity, but there were more variation hotspots (rps16_trnK-UUU, trnG-UCC_atpA, atpH_atpF, trnT-GGU_psbD, etc.) in the LSC region. By building evolutionary trees, it can be clearly observed that M. zhaojiaoensis formed a large group with eight species of Malus, but was relatively independent in differentiation. In conclusion, this study provides high-quality chloroplast genome resources of M. zhaojiaoensis and discusses the genetic variation characteristics of Malus genus. The findings of this study will provide a good reference for plastid genome assembly and interspecific comparison in the future.


Assuntos
Genoma de Cloroplastos , Malus , Códon/genética , Uso do Códon/genética , Genoma de Cloroplastos/genética , Filogenia
13.
Int J Biol Macromol ; 207: 927-940, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35364194

RESUMO

Up to now, unbalanced mechanical properties and poor heat resistance have become two major problems of polylactic acid (PLA). In this study, the coupling between Cellulose nanocrystal (CNC) and strong shearing field formed a unique hierarchical structure. Compared with pure PLA, the tensile strength of DPIM PLA/CNC increased from 57.9 MPa to 79.6 MPa without sacrificing the toughness of PLA, and the vicat softening temperature of DPIM PLA/CNC increased from 60 °C to 155 °C. The microstructure of PLA/CNC composites was analyzed by SEM, SAXS and WAXD, and it was found that the coupling effect of CNC and strong shear flow field could significantly change the crystallization behavior of PLA. CNC could increase PLA shish length from 251 nm to 889 nm under the action of shear field. At the same time, due to this coupling effect, more PLA shish-kebab structures were induced at the interface. This special hierarchical structure composed of CNC and PLA Shish-Kebab is of great significance and can provide important guidance for achieving the balance of strength and toughness of polymer materials.


Assuntos
Celulose , Nanopartículas , Celulose/química , Nanopartículas/química , Poliésteres/química , Espalhamento a Baixo Ângulo , Difração de Raios X
14.
Microb Biotechnol ; 2022 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-35298867

RESUMO

Phosphate-mobilizing bacteria (PMB) play a critical role in the regulation of phosphorus availability in the soil. The microbial genes pqqC and phoD encode pyrroloquinoline quinone synthase and bacterial alkaline phosphatase, respectively, which regulate inorganic and organic phosphorus mobilization, and are therefore used as PMB markers. We examined the effects of soil properties in three Moso bamboo forest sites on the PMB communities that were profiled using high-throughput sequencing. We observed differentiated responses of pqqC- and phoD-harbouring PMB communities to various soil conditions. There was significant variation among the sites in the diversity and structure of the phoD-harbouring community, which correlated with variation in phosphorus levels and non-capillary porosity; soil organic carbon and soil water content also affected the structure of the phoD-harbouring community. However, no significant difference in the diversity of pqqC-harbouring community was observed among different sites, while the structure of the pqqC-harbouring bacteria community was affected by soil organic carbon and soil total nitrogen, but not soil phosphorus levels. Overall, changes in soil conditions affected the phoD-harbouring community more than the pqqC-harbouring community. These findings provide a new insight to explore the effects of soil conditions on microbial communities that solubilize inorganic phosphate and mineralize organic phosphate.

15.
Acta Biomater ; 143: 295-309, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35301145

RESUMO

Vascularization of the poorly vascularized limbs affected by critical limb ischemia (CLI) is necessary to salvage the limbs and avoid amputation. Effective vascularization requires forming not only capillaries, but also arterioles and vessel branching. These processes rely on the survival, migration and morphogenesis of endothelial cells in the ischemic limbs. Yet endothelial cell functions are impaired by the upregulated TGFß. Herein, we developed an injectable hydrogel-based drug release system capable of delivering both VEGF and Dll4 to synergistically restore endothelial cellular functions, leading to accelerated formation of capillaries, arterioles and vessel branching. In vitro, the Dll4 and VEGF synergistically promoted the human arterial endothelial cell (HAEC) survival, migration, and formation of filopodial structure, lumens, and branches under the elevated TGFß1 condition mimicking that of the ischemic limbs. The synergistic effect was resulted from activating VEGFR2, Notch-1 and Erk1/2 signaling pathways. After delivering the Dll4 and VEGF via an injectable and thermosensitive hydrogel to the ischemic mouse hindlimbs, 95% of blood perfusion was restored at day 14, significantly higher than delivery of Dll4 or VEGF only. The released Dll4 and VEGF significantly increased density of capillaries and arterioles, vessel branching point density, and proliferating cell density. Besides, the delivery of Dll4 and VEGF stimulated skeletal muscle regeneration and improved muscle function. Overall, the developed hydrogel-based Dll4 and VEGF delivery system promoted ischemic limb vascularization and muscle regeneration. STATEMENT OF SIGNIFICANCE: Effective vascularization of the poorly vascularized limbs affected by critical limb ischemia (CLI) requires forming not only capillaries, but also arterioles and vessel branching. These processes rely on the survival, migration and morphogenesis of endothelial cells. Yet endothelial cell functions are impaired by the upregulated TGFß in the ischemic limbs. Herein, we developed an injectable hydrogel-based drug release system capable of delivering both VEGF and Dll4 to synergistically restore endothelial cell functions, leading to accelerated formation of capillaries, arterioles and vessel branching.


Assuntos
Capilares , Fator A de Crescimento do Endotélio Vascular , Animais , Arteríolas/metabolismo , Capilares/metabolismo , Células Endoteliais/metabolismo , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Isquemia , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/fisiologia , Regeneração , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
J Hazard Mater ; 432: 128736, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35339832

RESUMO

The widespread microplastics (MPs) pollution has become a concerning environmental issue. The interactions between MPs and typical pollutants may change the bioaccumulation, and toxicity of pollutants, leading to high uncertainty in risk assessment. Still, significant gaps remain in the knowledge available to integrate these interactions in the perspectives of toxicokinetics (TK) and toxicodynamics (TD), which is also an essential part of quantitative toxicological research. This review systematically summarizes the interaction between MPs and typical pollutants in TK and TD processes. MPs can be acted as the vector or sink of pollutants to increase or decrease their bioaccumulation, and also may not affect their bioaccumulation due to no interaction. The adverse outcome pathway (AOP) framework enables novel approaches for determining the interaction between MPs and pollutants in the TD process. MPs can directly or indirectly enhance, reduce and not affect the toxicity of pollutants. A series of factors influencing the interaction in TK and TD processes are summarized, including MPs characteristics and exposure scenarios. TK-TD approach can quantitatively understand the interaction between MPs and pollutants based on the mechanism. Given the current knowledge gap in TK and TD processes, future perspectives on combined exposure research are proposed.


Assuntos
Poluentes Ambientais , Poluentes Químicos da Água , Microplásticos/toxicidade , Plásticos/toxicidade , Toxicocinética , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade
17.
Nat Commun ; 13(1): 1396, 2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-35296675

RESUMO

The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
ACS Appl Mater Interfaces ; 14(8): 10344-10352, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35170946

RESUMO

The electrochemical CO2 reduction into formate acid over Pd-based catalysts under a wide potential window is a challenging task; CO poisoning commonly occurring on the vulnerable surface of Pd must be overcome. Herein, we designed a two-dimensional (2D) AuNP-in-PdNS electrocatalyst, in which the Au nanoparticles are intercalated in Pd nanosheets, for formate production under a wide potential window from -0.1 to -0.7 V versus a reversible hydrogen electrode. Based on the X-ray absorption spectra (XAS) characterizations, CO accumulation detection, and CO stripping voltammetry measurements, we observed that the intercalated Au nanoparticles could effectively avoid the CO formation and boost the formate production on the Pd nanosheet surface by regulating its electronic structure.

19.
Nat Commun ; 13(1): 891, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35173152

RESUMO

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.


Assuntos
Bioengenharia/métodos , Reatores Biológicos/microbiologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Prolina/biossíntese , Sequência de Bases , Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Edição de Genes/métodos , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Transporte Proteico/genética
20.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35105807

RESUMO

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Engenharia Genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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